Somatic hypermutation is a major mechanism for the diversification of immunoglobulin (lg) genes. After immunization, the somatic mutation process is activated in the precursors of memory B cells, apparently in the core of germinal centers in spleen, lymphnodes and other peripheral lymphoid organs. We have used transgenic mice carrying lg genes as a model to study somatic mutation and found that the mutations are targeted to the V-region and flanking sequences of lg genes. The following experiments are aimed at the unraveling of the molecular mechanisms which control this process: 1) Test substrates for somatic mutation were made which can be rescued from transgenic mice as plasmids and whose mutation results in easily scored changes in bacterial colony color. To overcome the inhibition of expression of the substrates, which is presumably due to the methylation of bacterial sequences within the gene, the existing mice will be bred to a modification-minus mouse strain and new transgenic mice will be produced with altered test genes. 2) Recent results have suggested that somatic mutation may require the binding of a mutator factor to a postulated DNA sequence, the mutation initiation region (MIR), upstream of variable (V) genes. In search for a MIR the upstream region of kappa-, lambda-, and H-genes will be sequenced. Transgenic mice will then be produced with test genes containing segments of DNA upstream of the promoter of V-genes, and as a control, upstream of the beta-actin gene. 3) Mutator positive B cells will be immortalized by fusion with a cell line which has been transfected with a mutation substrate. The substrate will then be rescued as plasmids and checked for mutations. 4) B cells derived from germinal centers will be cloned in an attempt to determine the cellular requirements for ongoing somatic mutation and its relationship to DNA replication. 5) PreB cell lines will be transfected with somatic mutation substrates and transferred into sublethally irradiated mice. Germinal center cells of the mice will be analyzed for somatic mutation of the test substrates. 6) Recent experiments have led us to a new model in which somatic hypermutation is linked to the direction of DNA replication. As a direct test of the model, transgenes will be produced in which an lg gene is flanked by an origin and a barrier of replication. Finding that mutation in transgenic mice only occurs when the lg gene is in one, but not the other orientation, would support the model. These experiments are hoped to lead to a clear picture of which DNA sequences in an lg gene are needed to initiate the mutation process, which sequences are dispensable, whether and in which direction DNA replication is required for the process and what accessory cells are involved. It is hoped that this work may aid in the treatment of diseases such as lymphomas where somatic hypermutation of lg genes presumably plays a role in the evasion of the malignant cells to treatment, as well as in the understanding of the pathogenesis of certain autoimmune diseases.